Competition between the replication initiator DnaA the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E-coli in vitro

Background: Following replication initiation, the replication origin (oriC) in Escherichia roll enters a hemimethylated state at Darn methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro, SeqA is essential for the synchronous initiation of chromosome replication front oriC copies in vivo. Results: We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC, (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small. hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. Conclusions: SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.